Project Summary/Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Even though most patients initially respond to platinum-based therapy, the likelihood of reoccurrence is virtually 100%. Clearly, an urgent need exists for developing tumor-selective therapeutics for EOC. One such option involves targeting EOCs via the folate receptor alpha (FR?) which is overexpressed in up to 90% of cases. C1 metabolism is critical to cancer cell survival and aberrant C1 metabolism in the mitochondria is frequently associated with malignancy. Current C1 inhibitors (e.g., pemetrexed) are plagued by problems of resistance and toxicity due to transport by the ubiquitously expressed reduced folate carrier (RFC). Mitochondrial C1 metabolism of serine beginning with serine hydroxymetyltransferase 2 (SHMT2) provides the majority of C1 units for biosynthesis of nucleotides in the cytosol and approximately 85% of endogenous glycine. Mitochondrial C1 metabolism also provides C1 donors required for translation of mitochondrial oxidative phosphorylation proteins and is critical to tumor metabolic adaption by protecting cells from hypoxia-induced cell death through production of NADPH and glutathione to maintain redox balance. There are no clinical inhibitors of SHMT2. We discovered a series of novel 5-substituted pyrrolo[3,2-d]pyrimidine analogs that inhibit mitochondrial C1 metabolism, specifically SHMT2, with secondary inhibition at cytosolic de novo purine biosynthesis, resulting in broad-spectrum antitumor activity. We propose to extend our studies to EOC including identification of next-generation analogs of this series with specificity for tumor-selective uptake by FR? and to refine our structure-activity-relationships for mitochondrial C1 targeting, leading to a new and highly selective therapy for EOC based on FR? expression and drug uptake. In aim 1, we will interrogate prospective inhibitors for FR?-specificity and drug efficacy in engineered Chinese hamster ovarian (CHO) cells which individually express the major transport systems (e.g. FR? and RFC) and cisplatin sensitive and resistant EOC cells expressing a range of FR?. Additional studies will measure plasma membrane transport properties for FR?-targeted lead analogs vis vis other transporters. In aim 2, we will determine the targeted pathways/enzymes of novel analogs in EOC through glycine/nucleoside ?rescue? from inhibition of cell proliferation, targeted metabolomics and studies with isolated enzymes targets. We will assay MFT uptake of lead analogs into mitochondria and measure mitochondrial oxidative stress and depolarization as a read-out of mitochondrial targeting. In aim 3, we will determine in vivo efficacies of the lead inhibitors of mitochondrial and cytosolic C1 metabolism in human EOC cell line and patient derived EOC xenografts in SCID mice. We will assess the impact of an intact immune system (including FR?-expressing tumor-associated macrophages) using a FR?- expressing syngeneic mouse model of EOC. If successful, our studies will provide ?first-in-class? inhibitors which target mitochondrial C1 metabolism with downstream effects on key cyotosolic C1 targets and are selective for FR? in ovarian cancer, including platinum resistant EOC.